Anti‑inflammatory compound curcumin and mesenchymal stem ... · 21 Anti‑inflammatory compound curcumin and mesenchymal stem cells in the treatment of spinal cord injury in rats

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Anti‑inflammatory compound curcumin and mesenchymal stem cells in the treatment

of spinal cord injury in rats

Jiri Ruzicka1,2†, Lucia Machova Urdzikova1†, Anna Kloudova1, Anubhav G. Amin3, Jana Vallova1,2, Sarka Kubinova1, Meic H. Schmidt3, Meena Jhanwar‑Uniyal3 and Pavla Jendelova1,2*

1 Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 2 Department of Neuroscience, Charles University, Second Faculty of Medicine, Prague, Czech Republic,

3 New York Medical College/Westchester Medical Center, Valhalla New York, USA, † These authors contributed equally to this work,

* Email: [email protected]

Spinal cord injury leads to a robust inflammatory response that is an unfavorable environment for stem cell implantation. In this study, we evaluated the effect of combined therapy of curcumin and mesenchymal stem cells (MSC) on behavioral recovery and tissue sparing, glial scar formation, axonal sprouting and inflammatory responses in a rat experimental model of spinal cord injury (SCI). Balloon‑induced compression lesion was performed at thoracic (Th8‑9) spinal level. Out of the four groups studied, two groups received curcumin on the surface of the spinal cord immediately after SCI and then once a week for 3 weeks together with an intraperitoneal daily curcumin injection for 28 days. The other two groups received saline. Seven days after SCI, human MSC were intrathecally implanted in one curcumin and one saline group. Both curcumin and curcumin combined with MSC treatment improved locomotor ability in comparison to the saline treated animals. The combined treatment group showed additional improvement in advanced locomotor performance. The combined therapy facilitated axonal sprouting, and modulated expression of pro‑regenerative factors and inflammatory responses, when compared to saline and single treatments. These results demonstrate that preconditioning with curcumin, prior to the MSC implantation could have a synergic effect in the treatment of experimental SCI.

Key words: spinal cord injury, human mesenchymal stem cells, curcumin, combined therapy, inflammatory response

INTRODUCTION

Spinal cord injury (SCI) leads to permanent deficits in sensory and locomotor functions below the injured spinal cord segment. The primary mechanical impact causes tis‑sue necrosis and rupture of neuronal and vascular struc‑tures (LaPlaca et al. 2007). As a consequence of the primary damage, secondary injury processes, including ischemia, glutamate excitotoxicity and local immunologic responses occur. The extent of secondary processes in incomplete in‑juries dictates the final severity of the injury. Thus, target‑ing these secondary processes is a potential opportunity

to reduce disability after the initial injury is sustained (An‑thony and Couch 2014, Lee et al. 2010, Wang et al. 2014a). Mesenchymal stem cells have shown their pro‑regenera‑tive and neuroprotective properties in several experimen‑tal models of SCI, as well as in models of Alzheimer’s dis‑ease and amyotrophic lateral sclerosis (Cocks et al. 2013, Forostyak et al. 2014, Kuroda et al. 2011, Ritfeld et al. 2012, Ruzicka et al. 2016). Their effect is attributed to paracrine action, mediated by growth factors, anti‑apoptotic and anti‑inflammatory molecules (Hawryluk et al. 2012, Teix‑eira et al. 2013). Worldwide, MSCs are already being used in clinical trials in the treatment of SCI and neurodegener‑ative diseases (Callera and do Nascimento 2006, Forostyak

Received 22 March 2018, accepted 5 December 2018

RESEARCH PAPER

Acta Neurobiol Exp 2018, 78: 358–374DOI: 10.21307/ane‑2018‑035

Combined therapy in treatment of SCI 359Acta Neurobiol Exp 2018, 78: 358–374

et al. 2013, Geffner et al. 2008). To facilitate MSC regener‑ative and protective potential, an appropriate combined therapy must be used to support MSC survival.

Curcumin also known as diferuloylmethane, (1E,6E)‑1,7‑Bis(4‑hydroxy‑3‑methoxyphenyl)hepta‑1,6‑diene‑3,5‑dione) is a well‑known anti‑inflammatory compound with described neuroprotective properties, which has already shown a positive effect on recovery after exper‑imental SCI (Machova Urdzikova et al. 2015, Ormond et al. 2012, Sanli et al. 2012, Wang et al. 2014b, Yuan et al. 2015). Following curcumin application, increased be‑havioral recovery after SCI has been observed (Macho‑va Urdzikova et al. 2015, Ormond et al. 2012, Zu et al. 2014). Curcumin is a natural inhibitor of major inflam‑matory cytokines such as tumor necrosis factor alpha (TNFα), interleukin 1 (IL‑1), IL‑8 and monocyte chemoat‑tractant protein 1 (MCP‑1) (Hidaka et al. 2002, Jain et al. 2009, Yuan et al. 2015). Curcumin application after SCI or TBI resulted in a suppressed inflammatory response via a TLR4‑MyD88‑NFkB dependent pathway, and thus indirectly affected the microglial activation and neuro‑nal apoptosis after injury (Ni et al. 2015, Zhu et al. 2014). The NFκB pathway has a strong correlation with TNFα and IL‑1 levels after injury (Yuan et al. 2015). Addition‑ally, curcumin affects STAT‑3 and NFκB pathways by in‑fluencing NO levels and reducing astrogliosis after SCI (Gokce et al. 2016, Machova Urdzikova et al. 2015, Sani‑varapu et al. 2016, Wang et al. 2014b). Moreover, cur‑cuminoids display the potential to decrease neuropathic pain via antagonizing TRPV1 channels (Lee et al. 2013). We hypothesize that the pre‑treatment of injured spinal cords with curcumin, prior to MSC implantation, might reduce the existing inflammatory environment, improv‑ing the survival of implanted cells and thus increasing the potential effect of the cell therapy.

The main objective of this study was to investigate whether the combined treatment of curcumin and MSC will have a synergistic effect in the treatment of the bal‑loon compression model of SCI. Locomotor and sensory recovery, spinal cord tissue preservation, glial scar for‑mation, axonal sprouting, relative expression of genes related to regenerative processes, and cytokine levels were evaluated, in both single and combined therapy, to determine the underlying mechanisms of the effect of curcumin, MSC, or their combination, on the develop‑ment of SCI.

METHODS

Animals

One hundred and thirty five Wistar rats (3 months old) were used for the purpose of this study. The opera‑

tion weight was set at 300 ± 15 g, in order to standardize the lesion size. All experiments were performed in ac‑cordance with the European Communities Council Direc‑tive of 22 September 2010 (2010/63/EU), regarding the use of animals in research and were approved by the Eth‑ics Committee of the Institute of Experimental Medicine ASCR, Prague, Czech Republic (approved under project number 35/2012).

As an experimental model of SCI, the balloon com‑pression lesion was used (Vanicky et al. 2001). Body tem‑perature was maintained at 37°C during the procedure. After induction of anesthesia (Isoflurane 300 mL/min, 3% Furane, AbVie, Prague, Czech Republic) a 2‑French Fogarty catheter was inserted into the epidural space (through the laminectomy of Th10), and positioned in the midline of Th8 segment. The balloon was inflated with 15 μL of saline for 5 min to induce severe compres‑sion injury. Animals received antibiotics (Gentamicine, 20 mg/mL, 50 μl i.m., Sandoz, Prague, Czech Republic) and painkillers (Rimadyl, 50 mg/mL, 50 μl i.m., Zoetis, Prague, Czech Republic) immediately after lesion in‑duction. Animals were randomly assigned to the four treatments groups; saline (n=34), curcumin (n=27), MSC (n=28), curcumin + MSC (n=26).

Animals receiving curcumin (curcumin, Sigma‑Al‑drich, St. Louis, MO, USA) were given a high dose once a week (60 mg/kg diluted in olive oil) intrathecally 4 times (immediately after SCI followed by 3 subsequent weeks), and a low dose intraperitoneally (6 mg/kg diluted in ol‑ive oil) daily, starting immediately after the injury and finishing on the 28th day after SCI. One week after SCI, the animals received intrathecal (area of L4‑5 segment) injections with either human bone marrow mesenchy‑mal stem cells (MSC, 5×105/50 μL) or saline (50 μL). Prior to and following the SCI, the first set of treated animals; saline (n=19), curcumin (n=12), MSC (n=13), curcumin + MSC (n=11) underwent a weekly series of behavioral and sensory tests. Nine weeks after SCI, the animals were sac‑rificed and histological, immunohistochemical and qPCR analyses were performed on spinal cord tissue (2cm long segment, with lesion area in the middle). Additional‑ly, the second set of animals were sacrificed at specific time points (10, 14 and 28 days after SCI) to determine the effect of the treatment on the immune response after SCI (n=5/group/time point). All treated animals were given daily i.p. doses of immunosuppression start‑ing three days before MSC injection until the end of the study (Cyclosporine A, 10 mg/kg, Sigma‑Aldrich, Germa‑ny) to prolong survival of the implanted stem cells.

The third set of animals was used to determine the survival of implanted stem cells at 3, 7, 14, 21 and 28 days after implantation (n=2 per group per time point). The last survival period was the end of the study (56 days post transplantation; Fig. 1).

360 J. Ruzicka et al. Acta Neurobiol Exp 2018, 78: 358–374

Behavioral testing

A series of locomotor and sensory functional tests were performed before SCI and then weekly for the en‑tire experimental period. All animals were precondi‑tioned and trained prior to the SCI.

BBB test

The open field basic locomotor ability was evaluated us‑ing the Basso, Beattie, Bresnahan (BBB) test scale (Basso et al. 1995). Two independent examiners studied the animal locomotor performance for 4 minutes, once a week, starting the week after SCI. Each hindlimb was evaluated separately. The average value of both hindlimbs was used for analysis.

Flat beam test

To evaluate advanced locomotor function, and, in par‑ticular, weight support and forelimb‑hindlimb coordina‑tion, the flat beam test with a modified Goldstein scale (Goldstein 1997) was used. The rats were scored from 0–7, ranging from no ability to balance on the beam, to crossing the whole length of the beam properly using both hind‑limbs. The test apparatus consisted of a 140 cm long device. The animals had to cross a central 1 m segment to reach a familiar darkened escape platform within 60 s intervals. This test was performed every second week for three con‑secutive days, with two trials per day. The average latency of performance was recorded using a video tracking sys‑tem (TSE‑Systems Inc., Bad Homburg, Germany)

Rotarod test

Highly advanced locomotor function including long term weight support and hindlimb coordination was

evaluated on four‑lane rotarod units (Carter et al. 2001) (Ugo Basile, Comercio, Italy), consisting of 7 cm diameter drums with grooves. As pre‑training, a 5‑minute interval of running exercise on an accelerating rod (5 rpm‑10 rpm) was performed daily for five consecutive days prior to the injury. Rats with SCI underwent four trials (each max 60 s) with a constant speed of 5 rpm in every testing session. The latency to fall from the rotating rod was automatical‑ly recorded. The test was performed before surgery and 3, 5, 7 and 9 weeks after surgery.

Plantar test

A standard Ugo Basile Apparatus (Carstens and Ans‑ley 1993) (Ugo Basile, Comercio, Italy) was used for the plantar (hot plate) test. The rats were always placed in separate semi‑transparent acrylic boxes, which protect‑ed them from watching each other, and a mobile device with an infrared heating lamp was positioned below the targeted hindlimb paw, always within the same region. Latency in withdrawal response to thermal nociceptive stimulus was automatically measured. Each paw was stimulated five times, with a stable pause‑measurement interval. The average value of both hindlimbs was used for analysis. Hyperalgesia was defined as a significant decrease in withdrawal latency. The latency of with‑drawal of a healthy trained individual is usually from 8 to 12 s. The hyperalgesia is below 50% of the latency of a healthy individual and was defined based on the single animal time curve comparison.

Stem cell culture and preparation

Human MSCs for this experiment were prepared un‑der good manufacturing practice (GMP) conditions and

Fig. 1. The flowchart shows the days of analysis after spinal cord injury (SCI; 0‑63) or human mesenchymal stem cell (hMSC; 0‑56) intrathecal (IT) administration. Curcumin was injected IP for 28 days consecutively and once a week IT starting at day 0 of the SCI. The scheme indicates time points of PCR, LUMINEX, histology and the behavioral part of the study. IHC‑ Immunohistochemical staining, IP‑ intraperitoneal, IT‑ Intrathecal, MSC‑ mesenchymal stem cells, SCI‑ Spinal cord injury.


supplied as a 1.5 mL cell suspension in Nunc tubes (Bio‑inova Ltd., Prague, Czech Republic; product name “Sus‑pension of Autologous MSC 3P”). Gradient centrifugation using Gelofusine (25%, B. Braun, Melsungen, Germany) was employed to separate the mononuclear fraction con‑taining MSCs from the bone marrow. The cell expansion was performed in media containing α Minimum Essen‑tial Medium (MEM) Eagle (without deoxyribonucleo‑tides, ribonucleotides and UltraGlutamin (Lonza, Basel, Switzerland)), supplemented with 5% mixed allogeneic thrombocyte lysate (BioInova, Prague, Czech Republic) and 10 μg/mL gentamicin (Lek Pharmaceuticals, Ljublan‑ja, Slovenia). For the purpose of the transplantation, cells from the second passage were analyzed and used. The expression of specific surface markers was detected with fluorescent‑activated cell sorting (FACS) analysis (FACSAria Flow Cytometer, BD Biosciences, San Diego, CA, USA). The cells were positive for CD90, CD105 and CD73 and negative for CD14, CD34, CD45 or CD79α, CD11b, and HLA‑DR (Human Leukocyte Antigens locus DR).

Tissue processing and immunohistochemistry

After finishing the behavioral experiment (i.e. nine weeks after SCI), the rats were transcardially per‑fused with paraformaldehyde (4% in PBS) and the spinal cords were post‑fixed overnight, removed from the spinal cord canals and transferred to PBS containing azide salt, from animals implanted with MSCs only, or MSC +curcum‑in, and several (n=2/ interval /group; 3, 7, 14, 21, 28 and 56 days after implantation) spinal cords were longitudi‑nally sectioned for the detection of implanted stem cells (Human Nuclei, HuNu, Millipore, Billerica, MA, USA). The rest of the spinal cords were dissected, 1 cm cranially and 1 cm caudally from the center of the lesion, mounted in paraffin, and cross‑sections (5 μm thickness) were cut. Sections were taken from 1‑mm intervals along the cra‑nio‑caudal axis from the center of the lesion. For the pur‑pose of the analysis, 15 cross sections, with the 8th cross section as the center of the lesion, were used. For analysis of tissue sparing, the square area of the whole cross sec‑tions was used. To distinguish the grey (GM) and white matter (WM), Cresyl violet (0.25 g of cresyl violet dis‑solved in 100 mL of distilled water with 1 mL of 10% acetic acid) and Luxol‑fast blue (1 g of Luxol‑fast blue dissolved in 100 mL of 96% ethanol with 5 mL of 10% acetic acid) staining was used (C5042 and L0294 both Sigma, St. Louis, MO, USA). The analysis of white and grey matter sparing, astrogliosis and the number of protoplasmic astrocytes after SCI, was calculated by the ZEISS AXIO Observer D1 microscope (Carl Zeiss, Weimar, Germany) and measured by Image J software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

Immunohistochemical analysis was performed to de‑tect microstructural changes after injury. The average μm2 areas of GAP43 positive fibers per mm2 were ana‑lyzed (n=5, 15 spinal cord cross sections/1mm interval). In order to detect axonal sprouting, a primary antibody against GAP43 (1:1000, AB5220, Millipore, Billerica, MA, USA) was used. As a secondary antibody, goat an‑ti‑mouse IgG conjugated with Alexa‑Fluor 488 (Abcam, Bristol, UK) was applied. The effect of the treatment on axonal sprouting was evaluated by LEICACTR6500 micro‑scope and TissueFax Software (TissueGnostics, Vienna, Austria). The graphical processing of the results was car‑ried out in Excel and CorelX6.

For the analysis of astrogliosis, the square area of the whole cross section was used. Primary antibody against GFAP (conjugated with CY3; 1:400, C9205, Sigma‑Aldrich, St. Louis, MO, USA) was used. The effect of the treatment on astrogliosis was analyzed with LEICACTR6500 micro‑scope. The percentage of glial scar per square area of the GFAP stained section and the number of protoplasmic astrocytes was determined by ImageJ software.

qPCR

To study the expression of selected rat genes related to the process of regeneration (Sort1/Nt3, Vegf, Bdnf, Ngf Casp3, Gfap, Gap43, Mrc1, Cd86, Irf5, CD163), the quantita‑tive real‑time reverse transcription polymerase chain reaction (qPCR), was used [for each group n=4, five sets of paraffin sections (interstitial tissue between IHC sec‑tions; each approx. of 500 μm spinal cord length; in‑cluding the central lesion]. RNA was isolated from para‑formaldehyde‑fixed sections using the High PureRNA Paraffin Kit (Roche, Penzberg, Germany), 9 weeks after SCI. The amount of RNA was quantified with a spectro‑photometer (NanoPhotometerTM P‑Class, Munchen, Germany). The isolated RNA was reverse transcribed into cDNA using Transcriptor Universal cDNA Mas‑ter (Roche, Penzberg, Germany), and a thermal cycler (T100™ Thermal Cycler, Bio‑Rad, Hercules, CA, USA). The cDNA solution, FastStart Universal Probe Master (Roche, Penzberg, Germany) and TaqMan® Gene Ex‑pression Assays (Life Technologies, Carlsbad, CA, USA) were used to perform the qPCR reaction. The qPCR was carried out in a final volume of 10 μl containing 25 ng of extracted RNA. StepOnePlus™ real‑time PCR cycler was used for amplification (Life Technologies, Carlsbad, CA, USA). All amplifications were run under the same cy‑cling conditions (2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C). Samples were tested in duplicate, and a negative control was included in each array. The relative quantification of gene expression was determined using the ΔΔCt meth‑


od (Pfaffl, 2001). Data was analyzed with StepOnePlus® software (Life Technologies, Carlsbad, CA, USA). The gene expression levels were normalized using Gapdh as a reference gene, and the values obtained from SCI rats treated with vehicle were set as zero. A log2 scale was used to display the symmetric magnitude for up‑ and down‑ regulated genes.

Luminex analysis

To analyze the effect of treatment on inflammatory response 10, 14 and 28 days after SCI, a Luminex assay was used (each group n=5 per time point). According to our previously published method (Urdzikova et al. 2014), 2 mm long spinal cord segments, dissected from the area of the injury epicenter were incubated in me‑dia for 24 h, and medium was then collected for further analysis. A customized Milliplex inflammatory cytokine kit (Millipore, Billerica, MA, USA) and Magpix instru‑mentation software were used to analyze levels of the inflammatory cytokines. Rat cytokine Luminex cus‑tom eight‑plex kit (for IL‑2, IL‑1β, IL‑4, IL‑6, RANTES, IL‑12p70, TNFα, MIP‑1α) was used for customized bead assays. According to the manufacturer’s protocol, the assays were performed in 96‑well filter bottom plates with antibody‑conjugated beads. The concentration of 5000 beads per marker was applied. To measure the levels of cytokines on a Luminex xMAP 200 system (Lu‑minex, Madison, WI, USA), biotinylated antibody with streptavidin‑RPE (streptavidin‑R‑Phycoerythrin; Life Technologies, Carlsbad, CA, USA) were used. Data were analyzed using Magpix software. The raw data [mean fluorescence intensity (MFI)] were used to calculate the concentration of each cytokine, using a five‑parameter logistic fit standard curve (generated for each cytokine from seven standards).

The lowest standard, which was at least three‑times above the background, was used to determine the low‑er limit of quantification (LLOQ). The LLOQ was calcu‑lated by subtracting the background (diluent) MFI from the MFI of the lowest standard concentration, and back calculating the concentration from the standard curve (Urdzikova et al. 2014). Data is expressed as %, where ‘healthy animal with no lesion’ is set as 100%.

Statistical analysis

Two‑way ANOVA was used to determine the level of statistical significance between treatment groups. In the case of behavioral tests (repeated measurements) or distribution of the effect (glial scar, white/grey mat‑ter sparing), two‑way repeated measurement ANOVA

was applied. As a post hoc pair‑to‑pair test, the Stu‑dent‑Newman‑Keuls (SNK) test was used (All Sigmastat 3.1 Sistat Software Inc., San Jose, CA, USA). Differences were considered statistically significant when p<0.05. Data in the graphs are shown as mean ± standard error of mean.

RESULTS

Cell survival after implantation

The survival of intrathecally implanted MSCs was visualized by antibody staining against human nuclei (HuNu). In the MSC group, the implanted cells were found up to 21days after treatment (representative im‑age 14 days, Fig. 2A), whereas in the case of the com‑bined therapy, the cells were found up to 28 days after implantation (representative image 21 days, Fig. 2B; survival histogram, Fig. 2C). In both groups, the im‑planted cells were found randomly distributed and attached to the surface of the spinal cord, no homing into the parenchyma was observed. Many cells were trapped in the folds of arachnoid mater at the site of the injection.

White and grey matter sparing

The effect of the treatment on the sparing of the white and grey matter was evaluated using cresyl‑vio‑let luxol fast blue stained serial cross sections. No sta‑tistically significant difference in white and grey matter sparing was observed between treatment groups. There was a trend toward significance in preservation of white and grey matter (within the cranio‑central region), when compared to saline treated animals. Both single treatments showed this effect prevalently in the white matter, whereas the combined curcumin + MSC treat‑ment supported the grey matter sparing. The caudal re‑gion of interest seemed relatively unaffected by any of the treatment applications (Fig. 3A ‑ A, B, representative image Fig. 3B, statistical values in Table I).

Structural changes in glial scar formation and GFAP+ cell morphology

The effect of the treatment on glial scar forma‑tion was measured on GFAP‑CY3 stained serial cross sections. No statistically significant effect of curcum‑in, MSC or combined treatment on downregulation of GFAP+ signal area was observed. In the distal parts of the measured area, predominantly in the cranial di‑


rection, both treatments containing curcumin slightly decreased the area of GFAP+. However, in the central region, the combined treatment, similarly to MSC ap‑plication, had the opposite effect. In addition, the num‑ber of protoplasmic astrocytes per section was count‑ed. When measured across the whole region of interest, no significant differences between groups were found. On the other hand, in the central region, MSC implant‑ed animals showed a statistically significant increase in the number of protoplasmic astrocytes, when com‑pared with saline or both curcumin containing treat‑ments. Additionally, curcumin + MSC treatment showed also significantly lower numbers in the central region, when compared to saline and curcumin treatment (central region: saline 24.3±3.6, curcumin 27.8±1.4, MSC

49.3±5.1, curcumin + MSC 9±4.2 (Fig. 3A–C, D, C1/D1, statistical values in Table I).

Axonal sprouting

Axonal sprouting was measured on GAP43‑stained serial cross sections. The average μm2 areas of GAP43 positive fi bers per mm2 were analyzed. A combination of curcumin and MSC signifi cantly increased the number of GAP43 positive fi bers, when compared to saline or both single treatments. Neither curcumin nor MSC single application had any eff ect on ax‑onal sprouting (saline=2.5×103±227, curcumin=3×103±114, MSC=2.1×103±279, curcumin + MSC=5.8×103±697) (Fig. 3A–E, E1, e1, 2, statistical values in Table I).

Fig. 2. The eff ect of curcumin application on the survival of implanted MSCs after SCI. The implantation of MSCs without curcumin led to 21 day survival of the grafted cells (representative image of spinal cord dorsal surface in close vicinity to lesion site, 14 days after SCI, (A). MSCs implanted within the period of curcumin treatment survived for 28 days after implantation (Representative image of spinal cord dorsal surface in close vicinity to lesion site, 21 days after SCI, B). The histogram showing the time dependence of MSC survival in curcumin/saline treated animals on the whole surface of 2 cm long segment of spinal cord with centralized cavity (C). Scale bar 100 μm, blue staining (DAPI), green staining (HuNu). Inserts show higher magnifi cation. Number (n) of animals per group (n=2/time point/group). ROI –Th7‑9.


Table I. Statistical evaluation of data from histology and imunohistochemistry analysis (Fig. 3), qPCR (Fig. 4) and behavioral tests (Fig. 6). First column; type of measurement and group identifiers. Second column; type of test applied (1WAN – one way ANOVA, 2WAN – two way ANOVA, 2WRMAN – two way repeated measurement ANOVA, post‑hoc tests SNK – Student‑Newman‑Keuls test, Dunn test). Third column; second factor of the analysis (time, section). Fourth column; group distribution test (F H or q values). Fifth column; p value.

Identifier Test used Factor B F/H/q test p value Identifier Test used Factor B F/H/q test p value

GM 2WRMAN 0.529 >0.05 Casp3 1WAN 5.078 <0.01

WM 2WRMAN 2.627 >0.05 SNK Inter CMSC vs. S 5.078 =0.01

S vs. CMSC SNK Inter 3Ca 3.469 <0.05 Bdnf 1WAN 2.071 >0.05

4Ca 4.112 <0.01 Ngf 1WAN 4.670 <0.05

MSC vs. CMSC SNK Inter 4Cr 3.948 <0.05 MSC vs. S SNK Inter 4.719 <0.01

3Ca 4.846 <0.01 MSC vs. C SNK Inter 4.666 <0.05

4Ca 4.115 <0.05 MSC vs. CMSC SNK Inter 3.276 <0.05

C vs. CMSC SNK Inter 4Ca 3.405 <0.05 BBB 2WRMAN 5.353 <0.01

Glial scar 2WRMAN 1.450 >0.05 C vs. S SNK Inter 5.331 <0.05

S vs. C SNK Inter Center 4.047 <0.05 1w SNK Inter 6.244 <0.001

1Ca 3.745 <0.05 6w SNK Inter 2.813 <0.05

2Ca 3.782 <0.05 FB test 2WRMAN 3.484 <0.05

MSC vs. CMSC SNK Inter 2Ca 3.323 <0.05 CMSC vs. S SNK Inter 4.440 <0.05

CMSC vs. C SNK Inter Center 3.498 <0.05 5w 4.151 <0.05

Morpho 2WRMAN 2.097 >0.05 6w 4.746 <0.01

MSC vs. S SNK Inter 5.062 <0.001 7w 5.048 <0.01

MSC vs. C SNK Inter 5.021 <0.001 8w 4.656 <0.01

MSC vs. CMSC SNK Inter 5.409 <0.01 9w 4.113 <0.05

S vs.CMSC SNK Inter 4.135 <0.05 C vs. S SNK Inter 3.161 >0.05

C vs. CMSC SNK Inter 3.792 <0.01 5w 3.954 <0.05

GAP43 1WAN 31.067 <0.001 6w 3.630 <0.05

MSC vs. S SNK Inter 12.277 <0.001 7w 3.468 <0.05

CMSC vs. C SNK Inter 8.701 <0.001 FB time 2.735 <0.05

CMSC vs. MSC SNK Inter 12.293 <0.001 CMSC vs. S SNK Inter 3.542 <0.05

qPCR M1/M2 3w 3.013 <0.05

Mrc1 1WAN 3.788 <0.05 4w 3.071 <0.05

MSC vs. S SNK Inter 3.518 =0.056 6w 3.880 <0.05

CMSC vs. S SNK Inter 3.678 <0.05 7w 3.547 <0.05

CD163 1WAN 14.438 <0.001 CMSC vs. C SNK Inter 3.035 <0.05

CMSC vs. S SNK Inter 4.322 <0.01 3w 3.563 <0.05

CMSC vs. C SNK Inter 8.673 <0.001 5w 3.071 <0.05

CMSC vs. MSC SNK Inter 5.644 <0.01 6w 3.038 <0.05

S vs.C SNK Inter 5.081 <0.01 Rotarod test 2WRMAN 2.010 >0.05

Irf5 1WAN 1.364 >0.05 C vs. S SNK Inter 3w 4.255 <0.05

CD86 1WAN 9.200 <0.05 C vs. CMSC SNK Inter 3w 4.674 <0.01

MSC vs. C Dunn Inter 2.963 <0.05 Plantar test 2WRMAN 1.941 >0.05

qPCR RAGs C vs. S SNK Inter 6w 4.208 <0.01

NT3 1WAN 0.097 >0.05 C vs. MSC SNK Inter 5w 3.790 <0.05

Vegf 1WAN 1.465 >0.05 6w 4.858 <0.01

Gfap 1WAN 1.354 >0.05 C vs. CMSC SNK Inter 6w 2.809 <0.05

Gap43 1WAN 8.304 <0.05


Expression of genes related to macrophage phenotype

The relative expression levels of genes related to M1 (CD86, Irf5) or M2 (CD163, Mrc1) macrophage phenotypes were measured 9 weeks after SCI. Curcumin application after

SCI slightly upregulated markers of both macrophage types, particularly Irf5 for M1, and CD163 for M2 phenotype. On the other hand, MSC or MSC + curcumin treatment either down‑regulated or did not change any of the monitored genes (Fig. 4A). In particular, none of the markers increased in the combined treatment group (statistical values in Table I).

Fig. 3A. Morphometric measurements of white (A) and grey (B) matter sparing showed no significant difference between treatment groups, with the predominant effect of the treatment in the cranio‑central region. Analysis of GFAP+ marker showed no significant difference in glial scaring (C), only in distribution of protoplasmic astrocytes in the MSC group (D). The effect of combined therapy on axonal sprouting, two months after SCI was observed (E). Statistical significance was marked with * when p<0.05. Number of animals per group (n=8/group). Representative images of IHC analysis (Astrogliosis after SCI‑ C1, Protoplasmic astrocytes‑ D1, (GFAP‑CY3), Axonal sprouting ‑E1, e1, e2, (GAP43)). The central, cranial and caudal region of GFAP morphometry measurement consists of 5 measured sections. Scale bars 200 μm (C1, E1), 30 μm (C1, e1, e2). Caudal‑X‑mm caudally to the injury site, Cranial‑X‑mm cranially to the injury site, n‑ number.


Table II. Statistical evaluations of data from cytokine evaluation (Fig. 5). First column; type of measurement and groups identifiers. Second column; type of test applied (1WAN – one way ANOVA, 2WAN – two way ANOVA, 2WRMAN – two way repeated measurement ANOVA, post‑hoc tests SNK – Student‑Newman‑Keuls test, Dunn test). Third column; second factor of the analysis (time, section). Fourth column; group distribution test (F H or q values). Fifth column; p value.

Identifier Test used Factor B F/H/q test p value Identifier Test used Factor B F/H/q test p value

Luminex assay C vs. S SNK Inter 3.166 <0.05

MIP1a 2WAN 4.662 <0.01 28D 3.555 <0.05

CMSC vs. S SNK Inter 3.347 <0.05 C vs. MSC SNK Inter 3.527 <0.05

14D 5.456 <0.001 28D 3.574 <0.05

CMSC vs. C SNK Inter 4.943 <0.01 IL12p70 2WAN 29.062 <0.001

14D 6.760 <0.001 CMSC vs. S SNK Inter 8.312 <0.001

CMSC vs. MSC SNK Inter 3.686 <0.05 10D 6.281 <0.001

14D 7.544 <0.001 14D 3.238 <0.05

IL4 2WAN 27.416 <0.001 28D 4.698 <0.01

CMSC vs. S SNK Inter 9.952 <0.001 CMSC vs. C SNK Inter 2.012 >0.05

10D 6.884 <0.001 28D 3.184 <0.05

14D 4.269 <0.01 CMSC vs. MSC SNK Inter 10.303 <0.001

28D 5.922 <0.001 10D 7.579 <0.001

CMSC vs. C SNK Inter 8.850 <0.001 14D 5.064 <0.01

10D 5.123 <0.001 28D 4.957 =0.001

14D 4.991 <0.01 C vs. S SNK Inter 8.149 <0.001

28D 5.296 <0.001 14D 5.408 =0.001


10D 9.952 <0.001 C vs. MSC SNK Inter 10.086 <0.001

14D 5.918 <0.001 14D 7.257 <0.001

28D 5.437 <0.001 28D 7.993 <0.001

IL1b 2WAN 13.329 <0.001 TNFa 2WAN 7.045 <0.001

CMSC vs. S SNK Inter 7.514 <0.001 CMSC vs. S SNK Inter 4.172 <0.01

10D 4.452 <0.01 10D 5.158 <0.01

14D 5.961 <0.001 CMSC vs. C SNK Inter 5.764 =0.001


14D 6.275 <0.001 CMSC vs. MSC SNK Inter 4.318 <0.05

CMSC vs. MSC SNK Inter 7.301 <0.001 RANTES 2WAN 23.346 <0.001

10D 5.529 <0.01 S vs. C SNK Inter 7.542 <0.001

14D 5.081 <0.001 10D 4.402 <0.01

IL2 2WAN 14.385 <0.001 14D 4.919 <0.01

CMSC vs. S SNK Inter 4.877 =0.001 28D 3.743 <0.05

14D 5.577 <0.001 S vs. CMSC SNK Inter 4.457 <0.01


14D 9.277 <0.001 MSC vs. S 3.019 <0.05

28D 4.824 <0.01 14D 3.162 <0.05

CMSC vs. MSC SNK Inter 6.289 <0.001 MSC vs. C SNK Inter 10.959 <0.001

14D 6.739 <0.001 10D 5.605 <0.001

IL6 2WAN 6.641 <0.001 14D 8.571 <0.001

CMSC vs. S SNK Inter 4.90 <0.01 28D 4.919 <0.01

14D 7.395 <0.001 MSC vs. CMSC SNK Inter 7.912 <0.001


14D 5.62 <0.001 14D 6.118 <0.001

28D 3.018 <0.05 CMSC vs. C SNK Inter 3.534 <0.05


14D 8.273 <0.001 28D 3.833 =0.01


Expression of genes related to regenerative processes

Selected genes from the host tissue for growth fac‑tors, apoptosis and astrogliosis were assessed 9 weeks after SCI using qPCR. The application of MSCs only, or

in combination with curcumin, significantly decreased the expression of Casp3 and increased the expression of Gap43. Differences were detected in the expression of Ngf, which was downregulated in the MSC only group. The curcumin application enhanced the expression of Nt3, Vegf, Bdnf and Gap43, and downregulated the expres‑

Fig. 3B. The representative images of white and grey matter sparing analysis in the cranial, central and caudal sections. Distal images are 7 mm from the lesion center. CVL – cresyl‑violet luxol. Scale bar 500 μm.


sion of Casp3, however, these changes were not statisti‑cally significant (Fig. 4B, statistical values in Table I).

Levels of proinfl ammatory cytokines

The effect of curcumin, MSC, or their combination on levels of proinflammatory cytokines in the center of the lesion was evaluated 10, 14 and 28 days after SCI, using a Luminex assay (Fig. 5A‑C). All three treatments

Fig. 4. Profi ling of relative expression of host genes related to macrophage response (A) and regenerative processes (B) in the lesion center aff ected by treatment, 2 months after SCI. Statistical signifi cance was marked with * when p<0.05. Number of animals per group (n=5/group). M1 indicate pro‑infl ammatory macrophage markers, while M2 are pro‑regenerative macrophage markers.

Fig. 5. Immune response to applied treatment 10 (A), 14 (B) and 28 (C) days after SCI. Ten days after SCI, a signifi cant increase in levels of IL4, IL1β, IL12p70 and TNFα, and a signifi cant decrease in RANTES were observed in the curcumin MSC group, when compared to saline as well as single treatment groups. Fourteen days after SCI, all cytokine levels, except for TNFα and RANTES, remained elevated in comparison with saline treated animals. Statistical signifi cance was marked with * when p<0.05. Number of animals per group (n=5/time point/group). No lesion ~ healthy animal with no lesion.


have shown a different impact on the levels of intrinsic immune molecules (MIP1α, IL4, IL1β, IL2, IL6, IL12p70, TNFα and RANTES). On the 10th day after SCI, the com‑bined treatment greatly increased levels of IL4, IL1β, IL12p70 and TNFα, and decreased levels of RANTES. On the other hand, MSC‑only treated animals showed low levels of cytokines, except for RANTES, which increased. These values obtained from MSC treated animals re‑mained unchanged for all the time points. On the 14th day after SCI, most of the inflammatory proteins (except TNFα and RANTES) remained elevated after combined treatment, when compared to saline treated animals as well as to both single treatment groups. The increase of

IL4, IL2 and IL12p70 also persisted until day 28. In cur‑cumin treated animals, increased levels of IL6, IL12p70 and decreased levels of RANTES were detected on day 14, and remained the same until the end of the experiment (day 28) (statistical values in Table I).

Locomotor and sensory recovery after SCI

Basic locomotor recovery was measured according to the BBB scale. Animals treated with curcumin, MSC or their combination showed increased locomotor recov‑ery after SCI, when compared to saline treated animals.

Fig. 6. Functional recovery after SCI. The locomotor performance of saline, MSC, curcumin or curcumin + MSC treated animals evaluated on BBB (A), Flat beam test (B, C) and Rotarod (D), 9 weeks after SCI. All treatments had a positive impact on locomotor recovery (A) after SCI. Combined therapy showed signifi cantly enhanced advanced locomotor skills (B, C). No hyperalgesia in response to nociceptive thermal stimulus on plantar test apparatus (E) was observed. Statistical signifi cance was marked with * when p<0.05. Number of animals per group (saline n=19, curcumin n=12, MSC n=13, curcumin + MSC n=11).


Statistical significance was observed only in the case of curcumin treated animals, in which a significant im‑provement was already observed from the first week af‑ter SCI. Only the curcumin + MSC group showed a strong trend towards significance (Fig. 6A, statistical values in Table I).

Advanced locomotor skills including weight support and forelimb‑hindlimb coordination were evaluated us‑ing the flat beam test. The flat beam test gives a more accurate view on the movement, coordination abili‑ty and balance so it can reveal more subtle differences than the BBB test. All treatment groups demonstrated at least moderately faster recovery when compared to sa‑line treated animals. However, only the curcumin + MSC combined therapy reached a statistically significant im‑provement in comparison with the saline group, when compared to all the data sets. The animals treated with curcumin scored significantly higher but only at weeks 5, 6 and 7, even though the curcumin showed a strong trend towards increased scores in the flat beam test as well as the time scores (Fig. 6C). Apart from the score, the time needed for crossing the selected region of the beam was measured. The shortest time in this task was achieved by animals from the curcumin + MSC group (Fig. 6B, statistical values in Table I).

To determine the effectiveness of treatment on the recovery of the high‑level locomotor score, long‑term weight support, endurance and hindlimb coordination, the rotarod test was used. As expected, taking into con‑sideration the severity of the injury model, none of the treatment groups significantly improved their perfor‑mance. Only curcumin treated animals at week 1 scored significantly better than controls and those which re‑ceived combined treatment (Fig. 6D, statistical values in Table I).

SCI leads to hyperalgesia. A plantar test apparatus was used to measure latency in response to painful ther‑mal stimulus (Ugo Basile, Comercio, Italy). No statisti‑cally significant change in the latency of response was observed in any of the treatment groups. In contrast, the tendency of curcumin and combined treatment to increase the latency of response, and thus attenu‑ate hyperalgesia has been observed. This tendency has reached, at several time points, statistical significance (Fig. 6E, statistical values in Table I).

DISCUSSION

Our study has demonstrated that the combination of the anti‑inflammatory compound curcumin and mesen‑chymal stem cells provides a mild synergistic effect in the treatment of spinal cord injury when compared to both single treatments, and shows enhanced recovery

when compared to vehicle treated animals. The com‑bined treatment affected MSC survival, endogenous in‑flammatory response, and axonal sprouting, which in turn led to a significant recovery in locomotor skills.

Both curcumin (Ormond et al. 2012, Zhang et al. 2017) and MSCs (Morita et al. 2016, Mukhamedshina et al. 2018, Nakajima et al. 2012, Urdzikova et al. 2006) were previously reported to show a positive effect on behavioral recovery after SCI. In our study, both sin‑gle treatments and their combination, had a positive impact on locomotor recovery after SCI. Curcumin had a major effect during the initial phase of the study, on both basic and advanced locomotor performance mea‑surements. Its immediate and continuous application after SCI, slowed down the secondary injury processes and thus accelerated and facilitated spontaneous re‑covery. Mesenchymal stem cell treated animals reached the same levels of BBB score, but over a longer period of time, showing only a mild impact on advanced lo‑comotor skills. A combination of curcumin and MSCs displayed no additional effect on the basic BBB test, however it showed the best flat beam test score, which assesses the advanced locomotor skills of the animals. The short‑term effect of MSC treatment may therefore correspond to their lower numbers at the end of the study, when the paracrine effect was not further pro‑longed. The effect of combined therapies on recovery after SCI is not always predictable, and described cases in the literature indicate that these therapies may not overcome the potential of single treatments, and may even bring undesired effects, such as delayed recovery or additional hypersensitivity of handicapped paws (Bretzner et al. 2008, Park et al. 2013).

The migration of MSC through cerebrospinal fluid into the injured spinal cord tissue was studied in Lew‑is rats. Penetration to spinal cord parenchyma and perivascular spaces, and MSC integration has been de‑scribed (Satake et al. 2004). Conversely, in our study, the MSCs persisted at the injection site in between the roots or were attached to the spinal surface. This might be due to the different model of injury, in which the dura mater is less affected in the region of the injury. However, in our study we observed the prolonged sur‑vival of MSC in combination with the curcumin appli‑cation. It was also noted that the cells were present in the spinal channel and since they did not home into parenchyma, their final numbers could have been be affected by tissue processing.

According to published studies, MSCs (Mukhamed‑shina et al. 2018, Ritfeld et al. 2012,) or curcumin (Or‑mond et al. 2012) showed a significant impact on white/grey matter sparing after SCI. In our study, a positive effect of co‑treatment was observed, but was not sta‑tistically significant. Their combination showed no


additional impact on total volume sparing, but a small positive effect in the area of the lesion epicenter was detected in the combined treatment. Both curcumin (Wang et al. 2014b, Yuan et al. 2015) and mesenchymal stem cells (Nakajima et al. 2012) have been described to attenuate the process of glial scar formation. In agree‑ment with previous findings, curcumin application showed a mild decrease of the total amount of GFAP+ area, similar to that effect observed in MSC treatment, but with a different pattern of signal distribution. In the case of the combination of these two treatments, no significant synergic decrease of GFAP+ area was de‑tected. However, in the curcumin group, and partic‑ularly in the combined therapy, the strongest effect on decreasing the number of protoplasmic astrocytes in injured spinal cord was observed. This could corre‑spond with findings that curcumin can have a positive effect on reactive astrocytes in various models of neu‑rodegenerative diseases or injured CNS (Daverey and Agrawal 2016, Jiang et al. 2011, Seyedzadeh et al. 2014, Wang et al. 2005). In contrast, in our study the MSCs led to the opposite effect, which is rather contradictory to what was shown in other studies using the applica‑tion of MSCs. This might be related to the lack of MSC penetration into the parenchyma in our study and thus a limited paracrine effect, when compared to the stud‑ies where the cells were directly implanted into the spinal tissue or migrated into the parenchyma (Liu et al. 2018, Park et al. 2015). The effect on astrocyte mor‑phology and function after SCI is one of the tools used to modulate the secondary processes after SCI, and mitigate the impact of the injury. Reactive astrocytes (A1 type) can produce cytotoxic factors and secrete cytokines to communicate with other glial cells; this then leads to decreased neuronal survival and plastici‑ty. The decreasing numbers of protoplasmic astrocytes after SCI can then promote axonal sprouting and sup‑port tissue sparing (Gaudet and Fonken 2018). In our study, a strong synergic effect on axonal sprouting was observed. Curcumin has a neuroprotective and anti‑in‑flammatory effect, which can increase the survival of neurons as well as glial cells leading to the preservation or recovery of myelin sheets (Naeimi et al. 2018, Zhao et al. 2017). Additionally, curcumin could have more impact on neurite outgrowth and axon protection (Lu et al. 2014, Ruzicka et al. 2018, Tegenge et al. 2014). In multiple studies, MSCs have been shown to support the axonal sprouting after SCI or TBI, mostly by paracrine action (Kocsis and Honmou 2012, Morita et al. 2016). The effect of curcumin application might be potentiat‑ed by the higher survival rate of MSCs in the combined therapy. In single MSC treatment, this effect might be transient and could slowly diminish at the time the tis‑sue is processed (i.e. two months after SCI).

The expression of selected genes related to endoge‑nous tissue regenerative processes in combined thera‑py exhibited an effect similar to MSC treatment alone. We observed downregulation of all macrophage related genes and caspase 3. All treatments increased the ex‑pression of the GAP43 gene, however, a significant in‑crease in the expression of GAP43 protein (increased axonal sprouting) was only observed in the combined treatment. This could be connected to MSC prolonged survival by co‑implantation with curcumin, and thus a longer impact of therapy on tissue reconstruction.

Despite this, curcumin only slightly upregulated all macrophage and several growth factor (Nt3, Vegf and Bdnf) genes. Our findings partially correspond to previ‑ous studies (Machova Urdzikova et al. 2015, Ormond et al. 2012, Urdzikova et al. 2014). On the other hand, both MSC and the combined group downregulated Casp3 expression, and combined therapy had a strong effect on the downregulation of macrophage related genes. In our previous studies, a single treatment with MSC (Ruzicka et al. 2017) or curcumin (Machova Urdzikova et al. 2016) has been shown to have a positive impact on increasing the M1/M2 ratio over time (10 and 28 days) following SCI. In our present study, we expected a sim‑ilar or more pronounced effect by the combined treat‑ment, which did not happen. We also performed a qPCR study 10 days after injury (3 days after cell application), however, we did not observe any differences between any of the animal groups (data not shown).

One of the mechanisms of secondary injury process‑es, leading to progressive damage on the cellular and spinal tissue levels, is inflammation. Several mediators including those tested in this study (MIP1α, IL4, IL1β, IL2, IL6, IL12p70, TNFα and RANTES) have been identi‑fied as playing a crucial role in this process. Curcumin has been shown to be a potent anti‑inflammatory com‑pound suppressing levels of IL1, IL8, MCP‑1 and TNFα across various disease models (Hidaka et al. 2002, Jain et al. 2009, Yuan et al. 2015). The application of cur‑cumin after SCI has been reported to show similar an‑ti‑inflammatory potential, by downregulating levels of inflammatory cytokines such as IL1‑β, RANTES, MIP‑1α and TNF‑α, and inhibiting several molecular pathways, including JAK/STAT, MAPK and nuclear factor‑κB acti‑vation (Chen et al. 2015, Dong et al. 2014, Lin et al. 2011, Zu et al. 2014). The application of MSCs displayed im‑munomodulatory potential by increasing levels of IL4 or IL13 and reducing TNF‑α, IL‑1 or IL6 levels (Nakajima et al. 2012, Tsumuraya et al. 2015). These findings are in agreement with the single therapies of MSC or curcum‑in in our previous studies (Machova Urdzikova et al. 2015, Urdzikova et al. 2014). However, in contrast, the combination of MSC and curcumin application showed not only an increase in levels of IL4, but also IL‑2, IL1‑β,


TNF‑α, and somewhat increased IL6 and IL12p70 lev‑els, when compared to saline treated animals and both single treatments. The reason behind the high IL4 lev‑els in the combined treatment could be an effect of curcumin, potentiated by MSC application (Machova Urdzikova et al. 2015, Ormond et al. 2012). On the oth‑er hand, proinflammatory markers such as IL‑2, IL1‑β, TNF‑α, and to some extent IL6 and IL12p70, are usually described to be suppressed by curcumin or MSC single application (Nakajima et al. 2012, Wang et al. 2014b). IL4 has been described as a pure anti‑inflammatory fac‑tor. Increased levels of IL4 after SCI have been shown to shift the microglia and macrophages response to the (M2) anti‑inflammatory and pro‑regenerative form (Francos‑Quijorna et al. 2016). IL6 was originally de‑scribed as a contributor to secondary pathological and inflammatory responses after SCI. However, recently it has been shown that increased doses of IL6 in myelin protein treated DRG cultures or an in vivo model of SCI can enhance axonal sprouting and decrease the levels of Nogo‑A or NgR. Moreover, treatment by IL6 can lead to functional recovery after SCI (Yang and Tang 2017). However, this imbalanced immune response during the combined treatment might be one of the reasons for the low synergy of curcumin and MSC implantation.

One of the major drawbacks of in vivo curcumin ap‑plication is its solubility in lipophilic substances. In our study, unmodified curcumin dissolved in oil was admin‑istered at the injury site in addition to systemic intra‑peritoneal application, in order to maintain bioavail‑ability for a period of at least 28 days. However, to ful‑ly support curcumin potential, a specific pH sensitive carrier (Requejo‑Aguilar et al. 2017) or modifications to increase solubility in water are needed.

CONCLUSIONS

The results of our recent study have confirmed only partial synergy in the treatment of experimental SCI with the combination of the anti‑inflammatory com‑pound curcumin and MSCs. The combined therapy led to significant preservation of advanced locomotor skills, and increased axonal sprouting, most likely due to the longer survival of the graft, modulation of the immune response, and changes in the expression of intrinsic genes related to regenerative processes after SCI. To potentiate the effect of preconditioning prior to stem cell implantation, the curcumin compound should be modified to fully release its potential in the injured spine region.

AUTHOR CONTRIBUTIONS

Pavla Jendelova, Lucia Machova Urdzikova and Jiri Ruzicka conceived the concept of the study, designed the experiments and wrote the manuscript. Anubhav Amin performed the cytokine analysis; Sarka Kubino‑va and Jana Dubisova performed the gene expression analysis and interpreted the data. All animal experi‑ments including SCI, behavioral testing, histology and immunohistochemistry were carried out by Jiri Ruzic‑ka, Anna Kloudova and Lucia Machova Urdzikova. Sarka Kubinova and Pavla Jendelova interpreted the data in relation to spinal SCI, while Meena Jhanwar‑Uniyal and Meic Schmidt interpreted the data in relation to cyto‑kine analysis.

ACKNOWLEDGMENTS

This study was supported grant GAČR P304/12/G069 and by European Union, the Operational Programme Re‑search, Development and Education in the framework of the project “Centre of Reconstructive Neuroscience”, registration number CZ.02.1.01/0.0./0.0/15_003/0000419 and by project InterAction LTAUSA17120. Pavla Jendelova, Lucia Machova Urdzikova, and Sarka Kubinova are mem‑bers of the project BIOCEV (CZ.1.05/1.1.00/02.0109) from the European Regional Development Fund, supported by LQ1604 National Sustainability Program II (Project BIOCEV‑FAR).

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Anti‑inflammatory compound curcumin and mesenchymal stem ... · 21 Anti‑inflammatory compound curcumin and mesenchymal stem cells in the treatment of spinal cord injury in rats